KR102627021B1 - PHARMACEUTICAL COMPOSITION FOR TREATING IMMUNE DISEASES COMPRISING MITOCHONDRIAl STAT3 EXPRESSION-INDUCED MITOCHONDRIA - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an immune disease comprising mitochondria isolated from zinc-treated stem cells as an active ingredient and a method for preventing or treating an immune disease using the same, which reduces pain and symptoms of osteoarthritis disease. It can alleviate and simultaneously regulate TH17 cells and Treg cells.

Description

Composition for treating immune diseases comprising mitochondria with induced mitochondrial STAT3 expression {PHARMACEUTICAL COMPOSITION FOR TREATING IMMUNE DISEASES COMPRISING MITOCHONDRIAl STAT3 EXPRESSION-INDUCED MITOCHONDRIA}

The present invention relates to a pharmaceutical composition for preventing or treating immune diseases containing zinc-treated stem cell-derived mitochondria as an active ingredient.

Although diseases caused by immune hypersensitivity reactions are increasing worldwide, the fundamental causes of these diseases have not been sufficiently identified. Currently, the treatment method for diseases caused by excessive immune responses is to alleviate or reduce various symptoms caused by the diseases by administering immunosuppressants alone or in combination.

Immunosuppressants refer to various substances used to reduce or block the host's ability to produce antibodies (humoral immune response) or cellular immune response in response to the action of an antigen. These immunosuppressants can be useful not only in the field of organ transplantation, but also in autoimmune diseases such as lupus and rheumatoid arthritis, and skin hypersensitivity reactions such as atopy and allergies. A good immunosuppressant must be able to control the imbalance of the immune response, ensure safety for the human body, and have a low incidence of disease recurrence during long-term treatment.

Immunosuppressants currently in use include cyclosporine A and FK506. These are natural compounds with complex chemical structures, which have the problem of high cost in terms of raw material supply, making them uneconomical, and long-term administration can cause various side effects. It contains risks. Therefore, there is an urgent need for the development of new immunosuppressants that can be economically produced with low toxicity and induction of immune tolerance.

Meanwhile, T cells are one of the cell groups that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body and go through a series of differentiation processes to differentiate into T cells with unique characteristics. T cells that have completed differentiation are broadly divided into type 1 helper cells (Th1) and type 2 helper cells according to their functions. Divided into accessory cells (Th2). Among these, the main function of Th1 cells is involved in cell-mediated immunity, and Th2 cells are involved in humoral immunity. In the immune system, these two cell groups maintain the balance of the immune system by checking each other to prevent each other from overactivating.

Therefore, most immune diseases can be seen as being caused by an imbalance between these two types of immune cells. For example, autoimmune diseases can occur when the activity of Th1 cells is abnormally increased, and when the activity of Th2 cells is abnormally increased. It is known that in this case, immune disease occurs due to hypersensitivity reaction.

Meanwhile, according to recent research results on the differentiation of Th1 cells, the existence of immunoregulatory T cells (Treg), a new group that can control the activity of Th1 cells, has become known, and research on the treatment of immune diseases using them is on the rise. , Treg cells have the characteristic of controlling inflammatory responses by suppressing the function of abnormally activated immune cells, and many studies are being conducted to treat immune diseases by increasing the activity of Treg cells.

In addition, in addition to Treg cells, another group created during the differentiation of T cells is Th17 cells. Th17 cells are known to be formed during the differentiation of undifferentiated T cells through a process similar to that of Treg cells. In other words, differentiation of Treg cells and Th17 cells is commonly accomplished in the presence of TGF-β, but Treg cells do not require IL-6, whereas Th17 cells require IL-6 along with TGF-β. Differentiation occurs in situations where Additionally, differentiated Th17 cells are characterized by secreting IL-17.

It has been revealed that, unlike Treg cells, Th17 cells are involved at the forefront of the inflammatory response seen in immune diseases and accelerate the progression of the disease by maximizing the signal of the inflammatory response. Therefore, in the case of autoimmune diseases that are not controlled by Treg cells, the development of treatments for autoimmune diseases targeting inhibition of Th17 cell activity is receiving great attention.

Currently, immunosuppressants that block the signal transduction pathway in T cells are most commonly used as treatments for immune diseases. These immunosuppressants cause toxicity, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, and nausea. There is a problem with side effects such as renal dysfunction.

In addition, in addition to treating immune diseases by suppressing the activation of T cells, treatments that control the amount of cytokines secreted from immune cells and treatments using antibodies targeting cytokines secreted from immune cells have been developed. It's in the middle. However, this method requires a lot of time to actually undergo clinical trials and apply to patients, and the method using antibodies has the problem of incurring too much cost in the antibody production process.

Korean Patent No. 10-1723265

In order to solve the above problem, the inventor of the present invention alleviated pain and symptoms of osteoarthritis disease by administering mitochondria isolated from zinc-treated mesenchymal stem cells (MSC) and simultaneously regulated TH17 cells and Treg cells. was confirmed and the present invention was completed.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating immune diseases comprising zinc-treated stem cell-derived mitochondria as an active ingredient, a method for preventing or treating immune diseases using the same, and zinc-treated stem cell-derived mitochondria. The purpose is to provide a manufacturing method.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating immune diseases containing zinc-treated stem cell-derived mitochondria as an active ingredient.

Additionally, the present invention provides a method for preventing or treating an immune disease comprising administering the composition for preventing or treating the immune disease to an individual in need of treatment.

Additionally, the present invention provides a method for producing zinc-treated stem cell-derived mitochondria, which includes culturing stem cells in a medium containing zinc and then isolating mitochondria from the culture.

The composition according to the present invention can relieve pain, relieve symptoms, and reduce joint damage of osteoarthritis. Additionally, the composition according to the present invention can effectively prevent and treat osteoarthritis by inhibiting inflammatory cell death of chondrocytes.

Figure 1 shows the results of measuring pain in an animal model of osteoarthritis caused by mitochondria derived from zinc-treated MSCs, according to an embodiment of the present invention.
Figure 2 is a CT analysis result confirming the ability to inhibit cartilage damage by zinc-treated MSC-derived mitochondria in an embodiment of the present invention.
Figure 3 shows the results of analysis confirming the ability of zinc-treated MSC-derived mitochondria to simultaneously regulate TH17/Treg cells in an example of the present invention.
Figure 4 shows the results of Western blotting analyzing the protein expression levels of cells and mitochondria in an example of the present invention.
Figure 5 is an analysis result confirming the effect of controlling inflammatory cell death in chondrocyte cells by administration of zinc-treated MSC in an example of the present invention.

Hereinafter, the present invention will be described in detail through embodiments of the present invention. However, the following embodiments are provided as examples of the present invention, and the present invention is not limited thereto, and the present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom. .

The present invention provides a pharmaceutical composition for preventing or treating immune diseases containing zinc-treated stem cell-derived mitochondria as an active ingredient.

The mitochondria may be isolated from mammals, including humans, or may be isolated from cells of surplus tissue, such as fat, of mammals, including humans. For example, the mitochondria may be isolated from one or more cells selected from the group consisting of somatic cells, germ cells, and stem cells of patients with immune diseases, such as osteoarthritis, but are not limited thereto. The stem cells may be one or more cells selected from the group consisting of mesenchymal stem cells, adult stem cells, pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue-derived stem cells, e.g. For example, it may be mesenchymal stem cells, but is not limited thereto.

In addition, mitochondria isolated from the patient with an immune disease can be injected to reduce the probability of immune rejection by injecting mitochondria from the patient with an immune disease, but the method is not limited to this.

The immune disease may include autoimmune disease or inflammatory disease.

In the present invention, the term "autoimmune disease" refers to the body's unresponsiveness to self-antigens as immunologic unresponsiveness or tolerance, and when problems arise in inducing or maintaining such self-tolerance, An immune response occurs against self-antigens, which causes an attack on one's own tissues. This refers to a disease caused by this process.

In the present invention, the term "inflammatory disease" refers to tumor necrosis factor-α (TNF-α), IL, which is secreted from immune cells such as macrophages by excessively promoting the human immune system due to harmful stimuli such as inflammatory factors or radiation. It refers to a disease caused by inflammatory substances (inflammatory cytokines) such as -1 (interleukin-1), IL-6, prostaglandin, luecotriene, or nitric oxide (NO). .

The above immune diseases include osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, spondyloarthropathy, inflammatory bowel disease, psoriatic arthritis, gout, bacterial arthritis, juvenile rheumatoid arthritis, lupus, scleroderma, multiple sclerosis, fibromyalgia, polymyositis, dermatomyositis, Behcet's disease, Reiter syndrome, Lyme arthritis, adhesive capsulitis, frozen shoulder, tendon synovitis, elbow capuchitis, DeQuervain tenosynovitis, recurrent rheumatoid arthritis, polymyalgia rheumatica, adult-onset Still's disease, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis. , asthma, primary cirrhosis, Goodfeitzer syndrome, autoimmune meningitis, Sjogren's syndrome, Addison's disease, alopecia areata, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes, dystrophic epidermolysis bullosa, epididymitis, glomeruli Nephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, sarcoidosis, scleroderma, spondyloarthrosis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, mitochondria. It may be one or more types selected from the group consisting of related syndromes and ulcerative colitis, for example, osteoarthritis, but is not limited thereto.

In the present invention, unless otherwise stated, "treatment" means reversing, alleviating, inhibiting the progression of, or preventing the disease or disease to which the term applies, or one or more symptoms of the disease or disease. means, and the term treatment as used herein refers to the act of treating when “treating” is defined as above. Accordingly, “treatment” or “therapy” of an immune disease in a mammal may include one or more of the following:

(1) Inhibiting the growth of immune diseases, i.e., arresting their development,

(2) preventing the spread of immune diseases, i.e. preventing metastasis;

(3) Alleviating immune diseases.

(4) preventing recurrence of immune diseases, and

(5) Alleviating symptoms of immune diseases (palliating).

The composition can prevent or treat immune diseases including osteoarthritis by inhibiting the activity of Th17, activating Tregs, and inhibiting inflammatory cell death of chondrocytes, but is not limited thereto.

The composition according to the present invention can be used as a pharmaceutical composition capable of preventing and treating immune diseases, and the pharmaceutical composition may further include a pharmaceutically acceptable carrier. In the above, “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., and those for parenteral administration such as water, suitable oils, saline solutions, aqueous glucose and glycols, etc. Carriers, etc., and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As for other pharmaceutically acceptable carriers, those described in the following literature may be referred to (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition according to the present invention can be formulated in a suitable form according to methods known in the art along with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various forms for parenteral or oral administration according to known methods, and a representative example of the formulation for parenteral administration is an isotonic aqueous solution or suspension for injection. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline solution or buffer solution and formulated for injection. Additionally, dosage forms for oral administration include powder, granules, tablets, pills, and capsules, but are not limited thereto.

Additionally, the present invention provides a method for preventing or treating an immune disease comprising administering the composition for preventing or treating the immune disease to an individual in need of treatment.

The treatment method of the present invention includes administering the composition for preventing or treating immune diseases to an individual in a therapeutically effective amount. The specific therapeutically effective amount for a specific subject will depend on the type and degree of response to be achieved, the specific composition, including whether other agents are used as the case may be, the subject's age, weight, general health, gender and diet, and time of administration. It is desirable to apply it differently depending on various factors including the route of administration, secretion rate of the composition, treatment period, drugs used together with or simultaneously with the specific composition, and similar factors well known in the medical field. Therefore, it is desirable to determine the effective amount of the composition suitable for the purpose of the present invention by considering the above-mentioned matters.

The subject is applicable to any mammal, and the mammal includes humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs and cats.

Additionally, the present invention provides a method for producing zinc-treated stem cell-derived mitochondria, which includes culturing stem cells in a medium containing zinc and then isolating mitochondria from the culture.

The zinc may increase COX4, which is related to the amount and function of mitochondria, in stem cell-derived mitochondria, and increase STAT3, which is known to enhance mitochondrial function and suppress apoptosis, but is not limited to this.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

[Example]

Example 1. Preparation of zinc-treated MSC-derived mitochondria

Fat tissue from an osteoarthritis patient was cut into small pieces, tissue by-products were filtered through a 100um filter, and centrifuged at 200g for 3 minutes at 4°C. Only the yellow fat floating on top was collected, and the tissue was separated into single cell units using collagenase. One hour later, 200g was centrifuged for 3 minutes at 4°C, washed leaving only the settled pellet, and the cells were separated using a 40um filter. The isolated cells were cultured in MEM medium containing 10% FBS. Zinc sulfide was treated by adding it to the cell medium at a concentration of 10nM for 3 days. After centrifuging the culture at 200g for 3 minutes at 4°C, the pellet was suspended in IBM buffer and the cell membrane was ruptured using a homogenizer. Then, centrifugation was performed using a mitochondrial isolation kit to obtain a mitochondrial pellet.

Example 2. Treatment effect of zinc-treated MSC-derived mitochondria on osteoarthritis

In order to confirm the effectiveness of zinc-treated MSC-derived mitochondria in treating osteoarthritis, the present inventors performed an experiment to analyze the treatment effect after injecting the vector into an animal model suffering from osteoarthritis.

After inducing MIA in the animal model, muscle-derived mitochondria isolated as in Example 1 (i.a) were injected into the joint cavity of the MIA model once a week at a concentration of 5ug/100ul one day later for a total of 3 weeks. Weight bearing was evaluated after MIA induction and was measured once a week.

As a result, when zinc-treated MSC-derived mitochondria were injected into animals with induced osteoarthritis, pain was reduced compared to the MIA group, an osteoarthritis-induced model, in both the first and second experiments, and pain was reduced more than that of MSC-derived mitochondria that were not treated with zinc. It was confirmed that it exhibited an excellent pain reduction effect (Figure 1).

Example 3. Protective effect on knee joint damage by zinc-treated MSC-derived mitochondria

In an animal model in which osteoarthritis was induced, it was confirmed through CT imaging whether joint damage was inhibited by zinc-treated MSC-derived mitochondria.

Figure 2 shows 4 of rats in which osteoarthritis was not induced (WT), a vehicle-treated group (Vehicle), an MSC-derived mitochondria-treated group without zinc treatment (MSC Mito), and a zinc-treated MSC-derived mitochondria-treated group (Zinc MSC Mito). Comparing the knee joints in the dog group using CT, it can be seen that the joints were almost undamaged in the WT group, but were significantly damaged in the excipient-treated group. On the other hand, the group administered zinc-treated MSC-derived mitochondria showed less damage compared to the excipient-treated group, and the damage was also less than that of the MSC-derived mitochondria-treated group that was not treated with zinc.

Example 4. Effect of zinc-treated MSC-derived mitochondria on TH17/Treg cells regulation

We analyzed whether TH17/Treg cells are controlled by zinc-treated MSC-derived mitochondria in an animal model in which osteoarthritis is induced. At 3 weeks after MIA induction, the rats were sacrificed, cells were isolated from the spleen tissue of each rat, and Th17 and Treg cell activities were analyzed by flow cytometry. For flow cytometry, CD4-PerCP, IL-17 APC, Foxp3-ECD, and CD25-FITC were stained, and the stained cells were analyzed by flow cytometry.

As a result, it was confirmed that the activity of inflammatory Th17 cells expressing IL-17 was reduced in the spleen tissue by intra-articular injection of zinc-treated MSC-derived mitochondria compared to untreated MSC-derived mitochondria (Figure 3). These results suggest that zinc-treated MSC-derived mitochondria can regulate not only joint inflammation but also systemic immune system pathogenesis.

Example 5. Confirmation of mitochondrial quantity and functional enhancement in zinc-treated MSCs

To confirm quantitative and qualitative improvement in mitochondrial function, protein expression levels of each cell and mitochondria were analyzed using the western bolt technique.

As a result, an increase in the COX4 gene related to the amount and function of mitochondria was confirmed in zinc-treated MSCs, and STAT3, which is known to enhance mitochondrial function and suppress apoptosis, was also confirmed to be increased. These results suggest that zinc treatment can increase the amount of mitochondria in cells and enhance their function.

Example 6. Confirmation of inhibition of inflammatory cell death of chondrocytes by administration to zinc-treated MSC-derived mitochondria

After stimulating 2x10 ⁴ cells of chondrocytes with Il-1b at a concentration of 10ng/ml, each MSC-derived mitochondria (5ug) was treated, the cells were cultured for 1 day, and cell death was detected through Annexin V/PI facs staining. was analyzed.

As a result, it was confirmed that MSC treated with zinc increased the survival rate of chondrocytes and suppressed necroptosis compared to MSC not treated with zinc.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (10)

A pharmaceutical composition for preventing or treating osteoarthritis comprising mitochondria derived from mesenchymal stem cells treated with zinc sulfide as an active ingredient,
The zinc increases the expression of COX4 and STAT3 in stem cell-derived mitochondria,
A composition wherein increasing the expression of COX4 and STAT3 increases the amount and function of mitochondria. The composition of claim 1, wherein the stem cells are patient-derived stem cells. delete delete The composition of claim 1, wherein the composition inhibits Th17 activity and activates Tregs. The composition of claim 1, wherein the composition inhibits inflammatory cell death of chondrocytes. delete delete delete delete